We propose to combine two rapidly evolving technologies to generate a single cell resolution atlas of the gene expression patterns that drive intestinal development. We have recently described a method for taking human induced pluripotent stem cells, exposing them to a series of growth factors, and directing the formation of stereotypical 3-dimensional intestinal organoids. Remarkably these show crypt-villus structure and the presence of a complete repertoire of differentiated cell types, as well as adult crypt stem cells. In addition we have recently developed a procedure, designated SCAMP, which allows accurate RNA-Seq analysis of the gene expression states of single cells. We propose to use RNA-Seq of single cells to capture the gene expression states that define the multiple developmental stages of human intestine development. Global transcriptomes will be determined for the starting human pluripotent stem cells, the induced endoderm, the primitive gut tube, and developing intestinal organoids. By comparing overlapping profiles it will be possible to precisely define gene expression progressions. By connecting the gene expression patterns that proceed from homogeneous intestinal gut tube cells, for example, to distinct intestinal cell types, and in particular the adult intestinal stem cell, it will be possible to preisely define each developmental program. To establish a base line for differentiated adult lineages we will also analyze single cells from crypts isolated from patient biopsies. We will then use this developmental atlas to interrogate intestinal organoids that we generate from induced pluripotent stem cell lines derived from a patient with intractable diarrhea of infancy syndrome (IDIS). This analysis will allow us to identify perturbations in normal developmental processes that may underlie the dysfunction in IDIS epithelium. In situ hybridizations and immunohistochemistry will be used to validate the RNA-Seq data, to localize cells with distinct gene expression profiles, and to reconstruct a 3-D image of the developmental process. In addition we propose to use a procedure we have established that allows a medium throughput analysis of the intestinal developmental function of genes. Gene knockdown is accomplished by using lentivirus to drive expression of shRNA in pluripotent stem cells. These are then induced to form organoids, and resulting developmental abnormalities examined. This procedure is at least ten times faster than can be accomplished with gene targeting in mice. The results of the proposed work would define the full molecular character of the multiple differentiated cell types of the adult human intestinal epithelium, as well the adult crypt stem cells. Novel molecular markers for these cells will be identified and complete molecular differentiation programs characterized. Genes will be functionally analyzed and genetic regulatory networks better defined.